DETERMINATION OF PHOSPHORYLATED AND SULFATED LINKAGE–REGION OLIGOSACCHARIDES IN CHONDROITIN/DERMATAN AND HEPARAN SULFATE PROTEOGLYCANS BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Abstract

The linkage-region oligosaccharides of chondroitin sulfate and dermatan sulfate, produced by the action of various chondroitinases, as well as of heparan sulfate, produced by the combined action of heparin-lyases I, II, and III, have been separated and characterized according to their size, number of sulfate residues, and the presence of phosphorylated xylose by HPLC. Glycosaminoglycans and/or proteoglycans were treated by tritiated borohydride and the [³H]-labeled xylitole-containing glycan chains were degraded by the various chondro/dermato-lyases (chondroitinases ABC, AC and B) and/or heparin-lyases. The produced linkage-region Δ-oligosaccharides have been completely separated by ion-pair reversed phase HPLC, using tetrabutylammonium as ion-pairing reagent, and detected by a radiochemical detector.

Application of the method to chondroitin sulfate and dermatan sulfate revealed that the first uronic acid after the -Gal-Gal-Xyl linkage trisaccharide is always glucuronic acid and the next galactosamine residue is sulfated. In porcine skin dermatan sulfate studied, a three glucuronic acid-containing cluster following the linkage-region oligosaccharide was found. None of chondroitin sulfate and dermatan sulfate studied contains phosphorylated xylose. This phosphorylation was, however, a dominating feature in xylose linkage-region of the glycans derived from rat chondrosarcoma proteoglycans.

The linkage region oligosaccharide fragment of heparan sulfate had the same structure and identical retention time with that obtained from dermatan sulfate with chondroitinase AC.

Acknowledgments